The long-term goal of my research program is to understand the molecular mechanisms that allow certain AB-type toxins to cross the endoplasmic reticulum (ER) membrane and enter the cytosol of an intoxicated eukaryotic cell. AB toxins consist of a catalytic A subunit and a cell-binding B subunit. A subset of AB toxins travels from the cell surface to the ER before A chain translocation into the cytosol. AB-type, ER- translocating toxins include cholera toxin (CT), pertussis toxin, Shiga toxin, and ricin. These toxins exploit the quality control mechanism of ER-associated degradation (ERAD) in order to move from the ER to the cytosol. Current models of toxin-ERAD interactions assume the toxin A chain is stable and protease- resistant, but recent work has shown that multiple ER-translocating toxins actually contain A chains that are thermally unstable after dissociation from the holotoxin. Based upon our work with the catalytic subunit of CT (CTA1), we have developed a new model of toxin-ERAD interactions in which toxin translocation, degradation, and activity are all linked to the heat-labile nature of the isolated toxin A chain. This model is in marked contrast to the prevailing view of ERAD-mediated toxin translocation and makes distinct predictions in regards to how host-toxin interactions affect the intoxication process. To test our model, this project will use a variety of biophysical and biochemical techniques to examine how the folding state of CTA1 affects, and is affected by, its association with components of the ERAD system and other eukaryotic factors known to interact with the toxin. Biophysical and biochemical studies of other ER-translocating toxins will also be used to test our prediction that thermal instability is a common property of toxins that exploit ERAD to enter the eukaryotic cell. The work of this project will produce a major conceptual shift in the pathogenesis of ER- translocating toxins, with direct applications to the development of new anti-toxin therapeutic strategies and to a basic understanding of the ERAD mechanism. RELEVANCE TO PUBLIC HEALTH: In order to attack the target cell, certain toxins must first unfold to enter the cell and must then refold inside the cell to become active. Factors associated with the target cell modulate this process, so an understanding of toxin-target interactions could lead to the development of novel anti-toxin therapeutics that prevent the unfolding and/or refolding events required for toxin activity.